Project Summary/Abstract Maintenance of body temperature at the optimal level is crucial for survival, and it requires homeostatic feedback regulation based on monitoring the temperature of internal organs as well as the environment. Homeostatic thermoregulation in response to changes of brain temperature relies on the temperature- sensitive neurons in the preoptic area of the anterior hypothalamus (POA). These neurons constitute about one third of the medial and lateral POA neurons and are intermingled with temperature-insensitive neurons that control drinking, feeding, sleep, and parental behaviors. For eight decades since the discovery of temperature-sensitive neurons in the brain, electrophysiology has been the only way to identify these central neurons. Having identified the first molecular marker for temperature-sensitive POA neurons by combining single-cell RNA-seq with whole-cell patch-clamp recording, we will identify central neurons that are upstream or downstream of these temperature-sensitive POA neurons, to elucidate the central neuronal circuitry for thermoregulation. To identify central neurons that receive input from temperature-sensitive POA neurons, we will use trans-synaptic tracers, and further verify these synaptic connections by using the PGDS Cre-line to drive channelrhodopsin expression in temperature-sensitive POA neurons for optogenetic activation in brain slices. To test whether specific neuronal types in the suprachiasmatic nucleus (SCN) innervate temperature- sensitive POA neurons to modulate the circadian variation of body temperature, we will use Cre-lines for these SCN neuronal types to drive trans-synaptic tracer expression. We will also use these Cre-lines to drive channelrhodopsin expression in SCN neurons, and record from POA neurons to determine whether they receive SCN input and whether their firing rate changes when the temperature of the brain slice is altered. In addition to identifying central neurons that are upstream or downstream of temperature-sensitive POA neurons, this proposed project includes mechanistic studies on the nature of the signals used by temperature-sensitive POA neurons to alter the activity of their downstream neurons so as to modulate body temperature, to test the hypothesis that, besides classical transmitters, endogenous PGD2 mediates thermoregulation. These studies will generate predictive models at a conceptual level of understanding thermoregulation.